scholarly journals Single occupancy spectroelectrochemistry of freely diffusing flavin mononucleotide in zero-dimensional nanophotonic structures

2015 ◽  
Vol 184 ◽  
pp. 101-115 ◽  
Author(s):  
Lawrence P. Zaino ◽  
Dane A. Grismer ◽  
Donghoon Han ◽  
Garrison M. Crouch ◽  
Paul W. Bohn
Keyword(s):  
Electron Transfer ◽  
Single Molecule ◽  
Light Emission ◽  
Fluorescence Emission ◽  
Flavin Mononucleotide ◽  
Wide Field ◽  
Potential Sweep ◽  
Potential Control ◽  
Fluorescence Measurements ◽  
Electron Transfer Properties

Zero-mode waveguides (ZMW) have the potential to be powerful confinement tools for studying electron transfer dynamics at single molecule occupancy conditions. Flavin mononucleotide contains an isoalloxazine chromophore, which is fluorescent in the oxidized state (FMN) while the reduced state (FMNH2) exhibits dramatically lower light emission, i.e. a dark-state. This allows fluorescence emission to report the redox state of single FMN molecules, an observation that has been used previously to study single electron transfer events in surface-immobilized flavins and flavoenzymes, e.g. sarcosine oxidase, by direct wide-field imaging of ZMW arrays. Single molecule electron transfer dynamics have now been extended to the study of freely diffusing molecules using fluorescence measurements of Au ZMWs under single occupancy conditions. The Au in the ZMW serves both as an optical cladding layer and as the working electrode for potential control, thereby accessing single molecule electron transfer dynamics at μM concentrations. Consistent with expectations, the probability of observing single reduced molecules increases as the potential is scanned negative, Eappl < Eeq, and the probability of observing emitting oxidized molecules increases at Eappl > Eeq. Different single molecules exhibit different electron transfer properties as reflected in the position of Eeq and the distribution of Eeq among a population of FMN molecules. Two types of actively-controlled electroluminescence experiments were used: chronofluorometry experiments, in which the potential is alternately stepped between oxidizing and reducing potentials, and cyclic potential sweep fluorescence experiments, analogous to cyclic voltammetry, these latter experiments exhibiting a dramatic scan rate dependence with the slowest scan rates showing distinct intermediate states that are stable over a range of potentials. These states are assigned to flavosemiquinone species that are stabilized in the special environment of the ZMW nanopore.

Electron Transfer in a Naphthalene Diimide System Studied by Single-Molecule Delayed Fluorescence

2020 ◽  
Vol 73 (8) ◽  
pp. 699
Author(s):  
Rosalind P. Cox ◽  
Saman Sandanayake ◽  
Steven J. Langford ◽  
Toby D. M. Bell
Keyword(s):  
Electron Transfer ◽  
Single Molecule ◽  
Delayed Fluorescence ◽  
Pmma Film ◽  
Time Resolved ◽  
Single Molecule Level ◽  
Film Interpretation ◽  
Fluorescence Measurements ◽  
Naphthalene Diimide ◽  
Prompt And Delayed Fluorescence

Electron transfer (ET) is a key chemical reaction in nature and has been extensively studied in bulk systems, but remains challenging to investigate at the single-molecule level. A previously reported naphthalene diimide (NDI)-based system (Higginbotham et al., Chem. Commun. 2013, 49, 5061–5063) displays delayed fluorescence with good quantum yield (~0.5) and long-lived (nanoseconds) prompt and delayed fluorescence lifetimes, providing an opportunity to interrogate the underlying ET processes in single molecules. Time-resolved single-molecule fluorescence measurements enabled forward and reverse ET rate constants to be calculated for 45 individual molecules embedded in poly(methylmethacrylate) (PMMA) film. Interpretation of the results within the framework of Marcus–Hush theory for ET demonstrates that variation in both the electronic coupling and the driving force for ET is occurring from molecule to molecule within the PMMA film and over time for individual molecules.

scholarly journals Approaches to single-molecule studies of metalloprotein electron transfer using scanning probe-based techniques

2017 ◽  
Vol 46 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Martin Elliott ◽  
D. Dafydd Jones
Keyword(s):  
Electron Transfer ◽  
Single Molecule ◽  
Research Area ◽  
Scanning Tunnelling ◽  
Single Molecule Studies ◽  
Tunnelling Microscopy ◽  
Active Research ◽  
Electron Transfer Properties ◽  
Transfer Properties ◽  
Active Research Area

The single-molecule properties of metalloproteins have provided an intensely active research area in recent years. This brief review covers some of the techniques used to prepare, measure and analyse the electron transfer properties of metalloproteins, concentrating on scanning tunnelling microscopy-based techniques and advances in attachment of proteins to electrodes.

Enhanced-Fluorescence of a Dye on DNA- assembled Gold Nano-Dimers Discriminated by Lifetime Correlation Spectroscopy

2017 ◽  
Author(s):  
Pedro M. R. Paulo ◽  
David Botequim ◽  
Agnieszka Jóskowiak ◽  
Sofia Martins ◽  
Duarte M. F. Prazeres ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Emission ◽  
Directed Assembly ◽  
Plasmon Mode ◽  
Correlation Spectroscopy ◽  
Enhanced Fluorescence ◽  
Detection Volume ◽  
Relaxation Component ◽  
Good Agreement ◽  
Confocal Detection

<div> <div> <div> <p>We have employed DNA-directed assembly to prepare dimers of gold nanoparticles and used their longitudinally coupled plasmon mode to enhance the fluorescence emission of an organic red-emitting dye, Atto-655. The plasmon- enhanced fluorescence of this dye using dimers of 80 nm particles was measured at single molecule detection level. The top enhancement factors were above 1000-fold in 71% of the dimers within a total of 32 dimers measured, and, in some cases, they reached almost 4000-fold, in good agreement with model simulations. Additionally, fluorescence lifetime correlation analysis enabled the separation of enhanced from non-enhanced emission simultaneously collected in our confocal detection volume. This approach allowed us to recover a short relaxation component exclusive to enhanced emission that is attributed to the interaction of the dye with DNA in the interparticle gaps. </p> </div> </div> </div>

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

scholarly journals Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription

2021 ◽  
Vol 22 (5) ◽  
pp. 2398
Author(s):  
Wooyoung Kang ◽  
Seungha Hwang ◽  
Jin Young Kang ◽  
Changwon Kang ◽  
Sungchul Hohng
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Facilitated Diffusion ◽  
One Dimensional ◽  
Bacterial Transcription ◽  
Fluorescence Measurements ◽  
Dimensional Diffusion ◽  
Single Molecule Studies ◽  
Hopping Diffusion

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

scholarly journals Low-Light Photodetectors for Fluorescence Microscopy

2021 ◽  
Vol 11 (6) ◽  
pp. 2773
Author(s):  
Hiroaki Yokota ◽  
Atsuhito Fukasawa ◽  
Minako Hirano ◽  
Toru Ide
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
Science Studies ◽  
Single Photon ◽  
Laser Scanning Microscopy ◽  
Oxide Semiconductor ◽  
Wide Field ◽  
Single Molecule Fluorescence ◽  
Low Light

Over the years, fluorescence microscopy has evolved and has become a necessary element of life science studies. Microscopy has elucidated biological processes in live cells and organisms, and also enabled tracking of biomolecules in real time. Development of highly sensitive photodetectors and light sources, in addition to the evolution of various illumination methods and fluorophores, has helped microscopy acquire single-molecule fluorescence sensitivity, enabling single-molecule fluorescence imaging and detection. Low-light photodetectors used in microscopy are classified into two categories: point photodetectors and wide-field photodetectors. Although point photodetectors, notably photomultiplier tubes (PMTs), have been commonly used in laser scanning microscopy (LSM) with a confocal illumination setup, wide-field photodetectors, such as electron-multiplying charge-coupled devices (EMCCDs) and scientific complementary metal-oxide-semiconductor (sCMOS) cameras have been used in fluorescence imaging. This review focuses on the former low-light point photodetectors and presents their fluorescence microscopy applications and recent progress. These photodetectors include conventional PMTs, single photon avalanche diodes (SPADs), hybrid photodetectors (HPDs), in addition to newly emerging photodetectors, such as silicon photomultipliers (SiPMs) (also known as multi-pixel photon counters (MPPCs)) and superconducting nanowire single photon detectors (SSPDs). In particular, this review shows distinctive features of HPD and application of HPD to wide-field single-molecule fluorescence detection.

scholarly journals Tunable Wide-Field Illumination and Single-Molecule Photoswitching with a Single MEMS Mirror

ACS Photonics ◽  
2021 ◽  
Vol 8 (9) ◽  
pp. 2728-2736
Author(s):  
Lucas Herdly ◽  
Paul Janin ◽  
Ralf Bauer ◽  
Sebastian van de Linde
Keyword(s):  
Single Molecule ◽  
Wide Field

The Single-Molecule Conductance and Electrochemical Electron-Transfer Rate Are Related by a Power Law

ACS Nano ◽  
2013 ◽  
Vol 7 (6) ◽  
pp. 5391-5401 ◽  
Author(s):  
Emil Wierzbinski ◽  
Ravindra Venkatramani ◽  
Kathryn L. Davis ◽  
Silvia Bezer ◽  
Jing Kong ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Single Molecule ◽  
Power Law ◽  
Transfer Rate ◽  
Electron Transfer Rate
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